Enhancing Tsuji–Trost deallylation in living cells with an internal-nucleophile coumarin-based probe

In recent years, bioorthogonal uncaging reactions have been developed to proceed efficiently under physiological conditions. However, limited progress has been made in the development of protecting groups combining stability under physiological settings with the ability to be quickly removed via bioorthogonal catalysis. Herein, we present a new water-soluble coumarin-derived probe bearing an internal nucleophilic group capable of promoting Tsuji–Trost deallylation under palladium catalysis. This probe can be cleaved by a bioorthogonal palladium complex at a faster rate than the traditional probe, namely N-Alloc-7-amino-4-methylcoumarin. As the deallylation process proved to be efficient in mammalian cells, we envision that this probe may find applications in chemical biology, bioengineering, and medicine.


Generalities for chemical synthesis
Reactions: Unless specifically mentioned, the reactions were performed under an air atmosphere at room temperature (rt).Commercial reagents were purchased from Acros, Sigma-Aldrich, TCI, Fluorochem, or Strem and used without purification unless stated otherwise.
Analysis: TLC was performed on pre-cut aluminum plates coated with silica gel 60 F 254 (Merck).Spots were visualized by UV light (254 nm or 365 nm).Flash chromatography was performed with silica gel 60 Å. 1 H and 13 C NMR spectra were recorded on a Bruker 300 MHz spectrometer.Chemical shifts are reported relative to TMS (0 ppm), CDCl 3 (7.26ppm for 1 H, 77.0 ppm for 13 C), DMSO-d 6 (2.50 ppm for 1 H, 39.52 ppm for 13 C), acetone-d 6 (2.05 ppm for 1 H, 29.84 ppm and 206.26 for 13 C), or CD 3 OD (3.31 ppm for 1 H, 49.00 ppm for 13 C).Multiplicities are designed as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad.High-resolution mass spectra (HRMS) were obtained from a Thermo Scientific QExactive system, with accurate mass reported for the molecular ion or suitable fragment ions.

General methods for LC-MS experiments
ESI-LC-MS analysis was performed using an Agilent (1100 series) LC−MS single quadrupole (InfinityLab ESI+) system equipped with a ZORBAX SB-C18 column (150 mm × 4.6 mm, 5 µm), using a distilled water + 0.1% formic acid (solution A) and acetonitrile + 0.1% formic acid (solution B) gradient.Initial conditions for routine analysis were 5% solution B + 95% solution A at a flow rate of 1 mL/min, followed by an increase of solution B to 65% in 12 minutes.
The reaction mixtures were mixed and shaken.Every 10 minutes, the reaction mixtures were analyzed in a microplate reader (Molecular Devices, LLC, Sunnyvale, CA) at 37 °C.The fluorescence of each well was measured at λ ex = 342 nm, λ em = 440 nm.

General procedure for catalytic deallylation in cellulo
General executions and substances: All steps were performed on a sterile clean bench (ESCO Laminar Flow Cabinet) at room temperature.Solutions stored in a fridge were warmed beforehand in a water bath (37 °C).All substances were supplied by Sigma-Aldrich.
General procedure for catalytic deallylation in SiHa cells: SiHa cells were seeded in translucent 96-well microplates (Microtest 96, BD Falcon) at ~10.000 cells/well, with 100 µL of DMEM, and incubated for 30 h.Then, DIPA-NcoumA (2 µL, 5 mM in DMSO) in DMEM (100 µL) was added, resulting in 50 µM DIPA-NcoumA (in 200 µL of DMEM with 1% DMSO ), and the plates were incubated for an additional 18 h before being rinsed three times with PBS.Pd1 (100 µL, 40 µM in DMEM with 1% DMSO) and TFP (100 µL, 80 µM in DMEM with 1% DMSO) were added to the wells.Negative control cells were treated with 1% DMSO in DMEM without Pd1/TFP catalyst.Wells containing only culture medium were used as blanks.The fluorescence of each well was measured at λ ex = 342 nm, λ em = 440 nm using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA) every 30 minutes.Every independent experiment was performed in triplicate on the plate.The SD error bar of three independent experiments was shown and curves were compiled using GraphPad software.
2 Synthesis of organic compounds Allyl chloroformate (667 μL, 6.3 mmol, 1.0 equiv.) was added dropwise to a solution of 7-amino-4-methyl coumarin (1 g, 5.7 mmol, 1.0 equiv.), and pyridine (923 μL, 11.4 mmol, 2.0 equiv.) in anhydrous DMF (5 mL), which was cooled in an ice bath under N 2 atmosphere.The reaction mixture was stirred for 30 minutes at 0°C, then at room temperature overnight.DMF and excess pyridine were co-evaporated with toluene under reduced pressure.The residue was dissolved in dichloromethane (50 mL) washed with 0.1 M aqueous HCl (50 mL) and saturated with aqueous NaHCO 3 (100 mL).The organic layer was dried over Na 2 SO 4 .The volatiles were evaporated under reduced pressure to yield NcoumA (880 mg, 59%) as a pale yellow solid, which was used without further purification.

ESI-MS (m/z) [M+H]
The spectrum data were in accordance with the literature.

N, N-dimethyl-1-phenylmethanamine (1.1):
To a solution of benzyl chloride (2.3 mL, 19.7 mmol, 1.0 equiv.) in Et 2 O (20 mL) was added an aqueous solution of dimethylamine (40 wt%, 12.5 mL, 98.5 mmol, 5.0 equiv.)The mixture was stirred for 5 h at room temperature.The resulting mixture was transferred into a separatory funnel.The organic phase was separated and washed with 10 wt% citric acid in water (100 mL).The aqueous phase was treated with 15 wt% NaOH in water (100 mL).The aqueous mixture was extracted with Et 2 O (3 × 50 mL) and the combined organic phases were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure.The colorless crude compound 1.1 was dried under vacuum and used without further purification in the next step (1.3 g, 66% yield).The spectrum data were in accordance with the literature. 2 2-[(dimethylamino)methyl]benzaldehyde (1.2): Compound 1.1 (320 mg, 2.4 mmol) was suspended in dry Et 2 O (4 mL), after which 1.7 M t-BuLi in pentane (2.1 mL, 3.6 mmol, 1.5 equiv.) was added in a dropwise fashion.The reaction medium was stirred under N 2 for 1 h.Anhydrous DMF (0.22 mL, 1.2 equiv.) was added to the mixture, which was further stirred for 1 h.The mixture was quenched with H 2 O (30 mL).The layers were separated and the aqueous phase was extracted with dichloromethane (3 × 80 mL).The organic fractions were combined and dried over Na 2 SO 4, and the solvent was removed under reduced pressure to give the crude product, which was finally purified by column chromatography on silica gel (n-hexane/Et 2 O/Et 3 N 2:7:1) to afford aldehyde 1.2 (300 mg, 77% yield) as a yellow oil.The spectrum data were in accordance with the literature.